MMG101 A103 Lab Practical I

prokaryotic

photoautotrophic

forms colonies

cyanobacteria

unicellular eukaryotic

heterotrophic

protozoa with pseudopod

unicellular eukaryotic

heterotrophic

protozoa with cilia

eukaryotic

saprophytic

multicellular with hyphae

prokaryotic, heterotrophic, nonmotile

Gram (+), purple, cocci clusters

known as Coagulase Positive Staphylococci

produces a variety of toxins, food poisoning, impetigo, wound infections

serious nosocomial pathogen

resistant to many antibiotics

MRSA

Thermal Resistance: >20 min

Catalase: (+)

Oxidase: N/A

TSA Plate: yellow pigment, insoluble

BAP: Beta-Hemolysis, clear halo

MAC: no growth of colonies

O/F Purple: (+/+) Fermentative, both yellow in closed and open tubes

virulence factors: coagulase enzyme

eukaryotic

heterotrophic

protozoa with flagella

eukaryotic in colonies, multicellular

photoautotrophic

algae with flagella

prokaryotic

Gram (+) purple rods

Thermal resistance: >20 min, least sensitive

Catalase: (+)

BAP: Beta-Hemolytic, tan halo

endospore former, hardy to UV irradiation

ubiquitous and contaminated frequently

Thioglycollate Broth Test: aerobe, milky growth on top of tube

prokaryotic, heterotrophic

Gram (+) purple, bacilli chains

prokaryotic

AUTO OR HETERO???

mobile- flagella

spiral-shaped bacteria

eukaryotic

saprophytic

unicellular or filamentous

found throughout the body in normal microflora: oral, GI, nasopharynx, opportunistic fungi, biofilm formation, budded to hyphal cell, fourth most common cause in nosocomial infections

creates germ tubes to reproduce strain, look like a tadpole

TJ: large, creamy opaque colonies

unicellular eukaryotic

photoautotrophic

protozoa/algae with flagella

Gram (-) pink bacilli in singles'

member of Enterobacteriaceae

in normal fecal flora, common reason for UTI in females

Thioglycollate Broth Test: facultative anaerobe, flocculated cloudy mixture

UV Irradiation: killed within >60 s.

Catalase: N/A

Oxidase: (-) no dot

TSA Plate: colorless with colorless medium, N/A solubility

BAP: Non-Hemolytic, no halo

MAC: Strong Lactose Fermenter, purple-pink colonies and purple medium

O/F Leifson: (+/+) Fermenter, bright yellow open and closed tube

O/F Purple: N/A

Gram (-) pink coccus singles

in a TSA slant filiform growth, no color

Thermal Resistance: >10 min

Gram (+) purple bacilli chains

in TSA plate, circular colony, entire, flat, opaque, dull and butyrous

color: bright yellow and turns agar yellow, thus WATER SOLUBLE

Gram (-) pink rods

in a TSB broth- no surface growth and diffused in general, no color

in TSA deep- complete growth on stab but no color

in TSA plate- confluent growth, no color change

in MAC- red colonies, beige medium

Thermal Resistance: >10 min.

Gram (+) cocci

Thioglycollate Broth Test: microaerophile, growth on top in a darker pink ring

Catalase: (+)

Oxidase: N/A

TSA Plate: yellow pigment, insoluble

BAP: no halo, non-hemolytic

MAC: no growth of colonies

O/F Purple: (-/-) Inert, purple in closed and open tube

O/F Leifson: N/A

Gram (+) cocci- clusters

Found in animals and humans normal fecal flora

opportunistic and nosocomial pathogen

VRE: Vancomysin Resistant Enterococci

indication of contaminated water via feces

Catalase (-)

Oxidative: N/A

TSA Plate: colorless with colorless medium

BAP: gamma, no halo

MAC: no growth of colonies

O/F Purple: (+/+) Fermentative open and closed tubes are both light yellow

O/F Leifson: N/A

can cause UTIs and endocarditis

virulence factors: hemolysins,

Gram (-) pink bacillus rod

produces pyocyanin (blue puss), water soluble

has grape-like smell

ubiquitous and loves water

associated with wounds, burns, and pneumonia

Catalase: N/A

Oxidase: (+) purple dot, VERY STRONG

TSA Plate: pyocyanin pigment, ocean blue, water soluble

BAP: Beta-Hemolysis, tan halo

MAC: Non-Lactose Fermenter but pigment producer, light purple/grey/beige colonies and green/gray medium

O/F Leifson: (+/-) Oxidative, yellow open tube and green closed tube

O/F Purple: N/A

Gram (-) pink rods, bacillus

anaerobe

hardy microbe, found in fecal flora and most common in feces

Thioglycollate Broth Test: anaerobe, growth on bottom of tube

Gram (+) purple cocci chains

Catalase: (-)

"pneumococcus"

BAP: Alpha-Hemolytic, green halo for partial lysis of rbc via hemolysin

neonatal sepsis, community acquired pneumonia

in normal throat flora

E-Test: MIC = 0.006-0.008 using antimicrobial ceftriaxome

has a capsule- increases virulence

obtain carbon from inorganic material, usually carbon dioxide, self-feeders, can be prokaryotic or eukaryotic

type of autotroph, use light as an energy source

ex: Anabaena, Volvox, Euglena

obtain carbon from organic material, such as CHO, fats, proteins, other organisms, can be prokaryotic or eukaryotic

ex: Amoeba, Paramecium, Lactobacillus, Typanosoma, Staphylococcus

organisms get their carbon compounds by living off dead, decaying organic matter

ex: most fungi

nitrogen fixation cells, synthesize their own CHOs, found in barren places, type of cyanobacteria

bacteria posses flagella, can be motile or non-motile

multiple flagella on a protozoa- flagellates

ex: Trypanosoma, Euglena, Volvox

only in eukaryotes, hair like structures, shorter than flagella, cover whole surface of microbe

ex: Paramecium

direction of cytoplasmic flow through a protrusion in the cell membrane

false foot, protrusion in the cell membrane that is formed by the movement of cytoplasm in the cell

ex: Amoeba

unicellular, smaller, larger than bacteria, reproduces by budding

rod-shaped bacteria

spherical-shaped bacteria

spiral-shaped bacteria

arise from spores, send out long filamentous structures which will elongate and branch out to create mycelium

multicellular, more filamentous, larger than bacteria, hypha

Eukaryotic: more complex internal organization with membrane-bound organelles, well-defined nucleus surrounded by a true membrane, no peptidoglycan in cell wall if wall is present, fungi, algae, plants, animals, protozoans

Prokaryotic: simpler, DNA is found but no true membrane-bound nucleus exists, no membrane-bound organelles, complex metabolic pathways, smaller and usually circular, peptidoglycan cell wall, bacteria and cyanobacteria

Start a the 10X objective and focus image in the center of the ocular lens. Rotate to 4X lens and add oil to the slide. Rotate 100X over slide and use fine focus knob to adjust and sharpen the image and increase light. Then, clean off the oil from the 100X objective and anywhere else on the microscope.

ring on the left ocular eyepiece that adjusts the focus of the left eye so differences in vision can be corrected

when focused and centered in a lower objective, the specimen image should be nearly focused in the next higher objective

dial that turns the light source on, intensifies the amount of light

resolving power of a microscope, ability to separate two points which are close together, smaller (d) resolving power, higher resolution

resolution = (wavelength of light) / (NA objective + NA condenser)

two lenses that bend the light entering so it is directed through the hole in the stage and concentrated at the level of the specimen

result of making an object appear larger than it really is, may not result in a clear image, first at the objective lens, than at the ocular lens level

opens and closes like a shutter to angle the amount of light hitting the slide, closing the shutter increases contrast but decreases resolving power.

use with 100X objective because not enough light when there is air space between the particular objective and the slide, creates a seal to allow more light into the objective resulting in greater resolution

ability of a medium to bend light rays

= objective power (4X,10X,40X,100X) x ocular power (10X)

expression of the light gathering capacity of the lens

(NA) = n sin (theta)

the distance between the objective lens and the specimen

shortens as the power of the objective increases

Calculate resolution = (wavelength of light)/(NA condenser + NA objective)

Magnification is making the image larger while resolution is making the image sharper/clearer

1. Ocular
2. Objective
3. Specimen Stage
4. Substage Condenser and Light Source
5. Focus Control
6. Base

Always use two hands, clean the lenses with Kimwipes, position 4X over stage when done, fold cord and tuck between stage and base, move condenser all the way down

100X immersion is best to use because it needs a higher amount of light and the oil immersion allows more light to be refracted on the specimen to see the image.

Adjust via rheostat, condenser or iris diaphragm

Make sure specimen slide is not upside down, is centered in the field of vision, increase brightness or refocus by changing the substage condenser height so its about 2 mm below slide, add oil if blurry or air bubbles.

has ocular eyepieces that magnify the specimen 10X, the diopter ring can be adjusted to focus the left eye so the differences in vision can be corrected

4X (red), 10X (yellow), 40X (blue), 100X (white), the working distance shortens as the power of the lens increases and surface area decreases

flat level stage and slide holder, use the X and Y knobs below the stage to move the slide

position of the condenser and control the amount of light going to the slide via the rheostat, the iris diaphragm, and condenser.

two co-axial focus control knobs that move the mechanical stage up and down, coarse focus knob and fine focus knob

supports the base of the microscope and contains the light source

a scale used to measure microorganisms with a microscope, small and large ocular units

4X = 25
10X = 10
40X = 2.5
100X = 1

= # small ocular units x correction factor

Mix culture if a bacteria, use Pasteur pipette to transfer culture to slide with 1 drop, put a cover slip on the liquid drop slowly as to not cause air bubbles and press on cover slide afterward.

Use minimal lighting

If you can't see anything, use less light by lowering condenser or closing iris down

If you see round, bright objects, move away from air bubble and look into a different spot

thin layer of cells immobilized onto a slide, barely visible to the naked eye, aseptically transfer cells to a slide using a sterile inoculating loop or a sterile Pasteur pipette

the immobilization of an organism and its structures by applying heat (physical method) or chemical agent, kills the cells quickly by denaturing their enzymes which stops metabolic activity that can alter staining and cause cells to adhere to the slide more readily

One goal is to get an even, thin distribution of cells

Minimizes distortion of cellular shape and spatial arrangement

Too many cells can prevent observation of the spatial arrangement and lead to decolorization

Two types are heat fixation and chemical agents

Heat: don't overcook bacteria, lose cellular integrity and characteristic morphology


FIX

1. wax circle and pass through flame to melt wax onto slide
2. sterilize loop
3. gently shake the tube to suspend microorganisms
4. pick up a loopful (2-3) of broth and put into wax circle and spread over circle or use a pipette to add 2-3 drops of the broth
5.fixation with heat on hot plate

1. wax circle and pass through flame to melt wax onto slide
2. put 1-2 drops of water into circle
3. sterilize loop
4. pick up a small amount of bacteria via needle and mix into drop of water *make sure it's not cloudy
5. fixation with heat on hot plate

If cell shape is distorted- shorten heat fixing time, allow loop to cool longer

If too thick and many cells layered on top of one another- suspend a smaller amount of sample into the water or slide

stains designed to separate bacteria into groups to facilitate the identification of them, use more than one dye and are used to distinguish among various groups of bacteria

cells retain primary crystal violet dye when complexed with iodine, appear purple-blue

cells are unable to retain crystal violet-iodine complex when treated with alcohol but will retain counterstain, safranin and appear red

Gram positive cells that sometimes stain Gram negative

high molecular weight repeating carbohydrate polymer linked by amino acid bridges, which forms the structural backbone of the cell wall

more in Gram (+) than Gram (-)

When underdecolorization occurs, cells appear purple when they should be pink, didn't use enough alcohol to decolorize sample

When overdecolorization occurs, cells appear pink when they should be purple, used too much alcohol to decolorize sample

leads to a false Gram negative, used too much alcohol to decolorize sample

leads to a false Gram positive, used too little alcohol to decolorize sample

shape of the bacteria

ex: bacillus, coccus, spirillum

crystal violet, enters both Gram (+) and (-) to stain deep purple

Gram's iodine, added after primary stain, makes the staining solution more intense, creates crystal violet-iodine complex

after mordant Iodine is added, treatment of stained cells with alcohol or acetone

removes lipids in Gram (-) cell walls, CV-I complex leaks from walls and cells become colorless

shrinks the Gram (+) walls trapping the CV-I complex so they still appear purple

Safranin, added after decolorizer alcohol, colorless Gram (-) cell walls take up Safranin and appear pink/red

Gram positive cells have more peptidoglycan and retain their CV-I complex while Gram negative cells have less peptidoglycan, leak their CV-I complex and retain counterstain, Safranin

Simple stain is a basic, one dye added to color cells while differential stains, like a Gram stain, differentiate between various types of bacteria in a sample.

The use of the decolorizer, alcohol can sometimes overdecolorize, leading to a false Gram (-) or underdecolorize, leading to a false Gram (+)

1. Gram stain reaction ((+) or (-))
2. Size (in microns)
3. Morphology (shape)
4. Spatial arrangement (singular v. colony)

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spherical- shaped bacteria

create diplococci, chains, tetrads, clusters

rod-shaped bacteria

create singles, pairs, chains

spiral-shaped bacteria

form spirilla (more rigid, helical) spirochetes (more flexible)

variety of shaped bacteria that don't fit in a specific category

1. Place smear-side up and add crystal violet for one minute.
2. Wash off with water and add Gram's Iodine and let sit for one minute.
3. Wash off with water and add 95% alcohol and use 1-2 drops per smear until the purple color no longer comes off in the alcohol.
4. Wash off with water and add safranin for two minutes.
5. Wash off with water and carefully blot slide dry.

Primary Step:
Gram (+) = purple
Gram (-) = purple

Mordant:
Gram (+) = purple
Gram (-) = purple

Decolorizer:
Gram (+) = purple
Gram (-) = colorless

Counterstain:
Gram (+) = purple
Gram (-) = red/pink/brown

Make sure there is enough oil on slide at 100X, make sure to blot slides carefully

If crystals present, make sure to wash off one stain completely before adding the next

If Gram (+) appears Gram (-), overdecolorization occurred. Make sure to use less alcohol during decolorization.

If Gram (-) appears Gram (+), underdecolorization occurred. Make sure to use more alcohol during decolorization.

If you cannot see single cells, remake smear to be one cell thick.

microorganisms are found in all natural environments

presence of a microbe in undesirable areas

the precautionary measures used to avoid contamination of cultures

When a hot loop is plunged into a liquid media because the microbe can be spread through aerosol formation through medium splatter.

dilution of bacteria by systematically streaking them over the agar surface in a petri dish to obtain isolated cells which will subsequently grow into piles of cells or isolated colonies.

useful to study colonial morphology, pigmentation, hemolysis.

piles of isolated cells

without viable microbial cells present

growth over the entire surface of the streaked area

if the agar surface grows microorganisms which are all the same, genetically.

dust particles, inanimate objects that can be combined with microbes and spread via air currents

used when a smooth lawn of growth over the entire plate of the agar is desired.

It's important to use aseptic techniques to avoid contamination of laboratory environment and microorganisms.

Dilution/isolation streaking techniques are important because you can study morphology, pigmentation, hemolysis and other characteristics of a single colony because it is isolated within a pure culture.

You get isolated colonies via dilution of the sample. A sterile loop of swab is used to obtain a microbial culture. It is streaked lightly. Sterilize the loop between streaks and fewer microbe colonies are deposited between the three zones.

Aerosol contamination is hazardous for everyone in the room because the microbes are now formed and spread through the air and can contaminate the laboratory.

We flame the loop to sterilize/inoculate the loop as to not spread contamination to the plate or into different zones.

We invert inoculated plates so condensation water drops do not drip down onto the surface of the plate and contaminate/dilute the microbes on the plate.

We leave the lids of petri dishes on at all times except when streaking or collecting a sample because you do not want to contaminate a sample with microbes from the air.

We do not place a contaminated loop on the bench because we don't want to contaminate the bench and spread the microbe elsewhere.

We don't place a hot loop in a liquid culture so as not to kill the microbes or contaminate the laboratory via aerosol formation.

We do not place a sterile loop on the bench during streaking because once placed on the bench, the sterile loop now becomes contaminated and must be resterilized.

Colony characteristics are observed on confluency plates while cellular characteristics are observed on dilution/isolation streaked plates, usually in Zone 3.

Contamination looks like anything that isn't your perceived results. Before plating, the agar surface should be smooth, moist, and free of anything growing in it. Keep an eye out for contamination (colonies growing), dehydration (cracked agar), or excessive moisture (droplets on surface of agar)

If there is no growth on the plate, allow loop to cool longer.

If there is no isolated colonies in zone 3, but there are in zone 2, increase number of zone to zone crossovers, make sure zone 1 is confluent or enlarge the zone 1.

If zone 3 shows confluent growth, make sure to sterilize loop between zones, use edge of loop to streak zones 2 and 3, make a smaller zone 1, decrease number of zone to zone crossovers.

If zone 1 has empty spaces, make sure you're using the flat side of the loop and increase number of streaks.

Gently shake tube and obtain a sample of liquid near the surface of the broth via loop or needle.

You obtain a small amount of growth on a sterilized loop.

You collect a sample from the outside edge of a well isolated colony.

You inoculate a liquid medium by touching sample to the surface of the broth, put on the cap and shake tube gently.

You inoculate a slant medium by starting at the base of the slant and zig zagging up the surface of the agar in a continuous motion.

You inoculate a deep medium by stabbing the sample with a needle into the deep medium until you are about 2 mm from the bottom. Pull needle out in the same path.

Using either the dilution/isolation streaking method or the confluency plate streak.

Human Body - 37°C
Room - 22°C
Fridge- 4°C
Hot Springs- >55°C
Arctic- <-20°C

1. Label Zone 1 on back of petri dish with necessary info.
2. Sterilize loop and obtain sample
3. Streak in small zone 1 for confluency, making sure to get corners and edges using the flat side of the loop. Start in the middle of zone 1 on the edge.
4. Sterilize the loop and cross over twice from zone 1 into zone 2 using the edge of the loop and making the streaks a little bit further apart.
5. Sterilize the loop and cross over once from zone 2 to zone 3 using the edge of the loop and making the streaks even further apart.

To streak for confluency, don't leave any gaps as you cover the whole plate in one direction with loop or sterile swab. Then turn the plate 90°, and cover the whole plate again in the one direction. Then turn the plate another 90° and cover the whole plate in one direction. Then get the edges of the plate before tossing the swab or sterilizing the loop.

A spread plate technique begins with putting the sample in the very middle of the plate. Sterilize the glass hockey stick with dirty ethanol and flame, then clean ethanol and flame. Once the spreader is cool, place the rod in the center of the agar plate and turn the turn table to move the sample around the plate, making sure to get the edges of the plate with the spreader as well. Resterilize hockey stick when completed.

no growth
ring growth
pellicle- covering over the top of the test tube

diffused- cannot see it well
flocculent- specks of contents in broth

surface growth- on top of stab line on surface

partial growth- some growth on surface and into stab line

complete growth on stab- growth on surface and all the way down the stab line

filiform- smooth even growth

beaded- many "beads" in growth pattern

rhizoid- feathery appearance

punctuate - <0.5 mm

circular

irregular-like a splash (Nickelodeon symbol)

rhizoid- feathery

filamentous- many branches

elliptical- oval shaped colony

entire- smooth edge

undulate- somewhat bumpy, uneven edge

lobate- like a splash (Nickelodeon symbol)

erose- fuzzy edge

flat- like a pancake

raised- raised on sides but flat in the middle

convex- like the coconut Patrick the Starfish lives under

pulvinate- more elongated, thinner convex elevation

umbonate- like a nipple

opalescent- opal, translucent and opaque

opaque- no light will pass through

translucent- light to pass through but no clear visibility

transparent- light passes through with clear visibility

dull or glossy

brittle- dry and crumbly

butyrous- butter like

membraneous- growth thin, coherent like membrane

viscid- growth that is wet looking

water soluble- color the solid medium

water insoluble- will not color the solid medium

**if microbe doesn't produce a pigment, solubility is N/A

round

oval

rhizoid- fluffy

filamentous- branched looking

entire- smooth edge

undulate- wavy uneven edge

erose- jagged sharp edge

feathery- filamentous

flat- like a pancake

convex- like a half circle

umbonate- like a nipple

raised- raised on edge but flat in the middle

filamentous- like hairs or fibers

folded- like gathered material

furrowed- deep grooves and wrinkles

glaborous- smooth surface

rugate- folded and wrinkled like stomach lining

wrinkled- like brain tissue

cottony-like cotton fibers

downy- like fine, soft feathers

fluffy- soft, loose light airy mass

granular- grainy appearance

powdery- crumbly looking

velvety- smooth like silk

waxy- smooth soft pliable surface like dried wax

check on surface and reverse (through the agar) side, two different colors can be obtained

Aerial hyphae

proteins that catalyze biochemical reactions in pathways, make reactions proceed quickly and efficiently (catalysis)

building cellular macromolecules from nutrients

breaking down macromolecules for energy or conversion, most reactions are catabolic

designed to pose nutritional or metabolic questions to a pure culture under study.

Part of biochemical test media, one of two ways: What kind of organic compounds can the microbe use as energy source, supplying a large amount a organic compounds and determining if they were utilized or not.

What end-products or by-products does the microbe produce as the result of enzymatic modification, excretion of waste, detected by pH indicators

usually dyes that colorimetrically determine what wastes or organic acids have accumulated in the media.

used to grow a wide variety of microbes, variety of nutrients to support many bacteria with different nutritional requirements, commonly used for isolating microbes from a mixed culture and maintain pure bacterial cultures.

permit the growth of certain bacteria while preventing the growth of other bacteria

ex: high salt conc., dyes, inhibitory chemicals, antibiotics

allow a distinction between two species or among groups of microbes by determining the presence of absence of specific enzymatic pathways

ex: chemical or cellular indicator

General Nutritional Media sponsors almost all growth and nutritional needs on a plate. Selective media only permits the growth of some bacteria. Differential media allow distinction between two species with specific enzymatic pathways.

How Does This Work?: peptones used for various nutritional requirements for many microbes
Original Color of Medium: beige/tan, translucent, N/A
Microbes Selected For and Against: used for phenotypic testing and pigment production
Selective Agents (Inhibitors): N/A
Differential Agents: N/A
pH Indicator: N/A
Interpretation of Results:
TSA slant- growth patterns on slant media
TSA deep- oxygen requirements of microbes
TSA plates- isolation, usually dilution/isolation streak but can streak for confluency for endospore production

How Does This Work?: identify with hemolytic action (hemolysins) on red blood cells in medium, dilution/isolation streak and stab media twice, incubated in 37°C CO2

Original Color of Medium: opaque red

Microbes Selected For and Against: for nutritionally fastidious organisms, identification of streptococci

Selective Agents (Inhibitors): N/A

Differential Agents: highly differential medium, sheep's red blood cells

pH indicator: N/A

Interpretation of Results:
for streptococcal microbe:

A- Hemolysis- green halo around colony, partial lysis of rbc
B-Hemolysis- tan halo around colony, complete lysis of rbc
Gamma- Hemolysis- no discoloration of medium, not hemolysins active against rbc
A'-Hemolysis- double zone, some rbc are intact while others are completely lysed

for all other microbes:
B- Hemolysis- clear, tan halo around colony, complete lysis of cell by hemolysin
Non-Hemolytic- no tan or green halo is seen, no hemolysins active against rbc

How Does This Work?: allow hardy Gram (-) to grow and to determine if they are lactose metabolizers (fermenters) or not (non-fermenters), dilution/isolation streak incubate at 37°C O2

Original Color of Medium: light purple

Microbes Selected For and Against: aid in the isolation of Salmonella and Shigella species from fecal flora

Selective Agents (Inhibitors): crystal violet and bile salts, inhibit growth of Gram (+) and fastidious Gram (-) cells, allow hardy Gram (-) to grow

Differential Agents: lactose, separate microbes who can metabolize lactose with acidic end products (fermenters) or cannot use lactose, alkaline end-products (non-fermenters)

pH Indicator: neutral red, fermenters = red, non-fermenters = beige

Interpretation of Results:
strong lactose fermenter- colonies and surrounding medium are purple-pink/red, contain permease and beta-galactosidase

weak lactose fermenter- colonies are pink with dark red center, bullseye from bottom of petri dish, but pearly pink from above, limited permease but has beta-galactosidase

non-lactose fermenter- beige colonies and medium, may be grey-purple

non-lactose fermenter but pigment producer:
red colonies, beige medium = Serratia marcescens
light purple/grey/beige colonies, green/gray medium = Pseudomonas aeruginosas

How Does This Work?: determines microbe's mode of utilization of a CHO, oxidation of glucose, low conc. of peptone, one base tube (no glucose), two glucose tubes, stab, one has mineral oil (closed tube), no mineral oil (open), 37°C O2

Original Color of Medium: blue-green

Microbes Selected For and Against: used with Gram (-) rods only

Selective Agents (Inhibitors): N/A

Differential Agents: glucose

pH Indicator: bromothymol blue, turns canary yellow under acidic conditions, neutral it's green, alkaline it's blue

Interpretation of Results:
Oxidative (+/-) = yellow on top of open, green in closed
Fermentative (+/+) = yellow on open, yellow on closed
Inert (-/-) = green on open, green on closed

How Does This Work?: test Gram (+) microbes, purple medium more nutrients for Gram (+), use peptones if not using glucose

Original Color of Medium: Purple

Microbes Selected For and Against: used for Gram (+) microbes only

Selective Agents (Inhibitors): N/A

Differential Agents: glucose

pH Indicator: bromocresol purple

Interpretation of Results:
Oxidative (+/-) = light yellow on open, purple on closed
Fermentative (+/+)= light yellow on open, light yellow on closed
Inert (-/-) = purple on open, purple on closed

How Does This Work?: fresh sample to a slide, add 3% Hydrogen Peroxide and look for bubble formation, looks for catalase enzyme that turns h202 into water and oxygen

Microbes Selected For and Against: used for Gram (+) microbes, distinguishing between streptococci and staphylococci

Interpretation of Results: if bubbles form = catalase enzyme is present, no bubbles = no catalase enzyme

How Does This Work?: used to determine presence cytochrome oxidase, present in most aerobic bacteria

Original Color of Medium: Dry-Slide oxidation

Microbes Selected For and Against: Gram (-) microbes

Interpretation of Results:
Oxidase (+) = turns purple within 20 seconds
Oxidase (-) = sample on card remains the same color, turns purple after 20 seconds

We need to perform biochemical test because staining and characteristics are not sufficient to identify bacteria because there are simply too few characteristics to identify more than 13,000 types of classified bacteria thus far.

So as to not have contamination within results of media testing.

Catalase test

Oxidase test

How Does This Work?: ability to produce a neutral end product from glucose fermentation, detects acetoin formed during metabolization of glucose, add KOH and alpha-naphthol

Original Color of Medium: N/A

Microbes Selected For and Against: for butylene-glycol fermenters

Selective Agents (Inhibitors):

Differential Agents:

pH Indicator:

Interpretation of Results:
Positive VP: pink ring forms at top of tube, ferments glucose via butylene glycol pathway, ex: Klebsiella and enterobacter genus

Negative VP: no ring forms, mixed acid fermenter ex: E. coli and Citrobacter species

microbes that utilize light as their energy source

microbes that require a chemical energy source

microbes that utilize carbon dioxide as their energy source

microbes that utilize organic carbon as their energy source

microbes that utilize light as their energy source and use carbon dioxide as their carbon source

microbes that utilize light as their energy source and use organic carbon as their carbon source

microbes that utilize a chemical energy source and atmospheric carbon dioxide as their carbon source

microbes that utilize a chemical energy source and an organic source of carbon

readily supplied nutrients in large quantities, never occur in free elemental form but in compounds, these are C, N, S, K, Mg, Ca, Na, P

required in trace amounts and are frequently supplied in contaminants of major nutrients, from the air or leached from the glass of the medium, these are Mn, Fe, Co, Cu, Zn, Mb

CHO, Alcohol, Peptones and proteins, amino acids, carbon dioxide

inorganic forms of nitrogen like ammonia, and ammonium salts, atmospheric nitrogen, nitrogen in amino acids

sulfates in inorganic salts, organic bound sulfur, sulfur in amino acids, hydrogen sulfide

medium that has the minimal nutritional requirements for a chemoheterotrophic organism to grow, like E. coli on glucose and inorganic salts

microbe that can live in a low nutrient environment

microbe that needs numerous nutrients to live in the environment, sometimes nutrients must be added for microbe to grow like Streptococci

microbe between hardy and fastidious with respect to nutritional requirement.

minimum temperature for an organism to grow

maximum temperature an organism can grow in

temperature that allows for best growth, growth is most rapid because chemical and enzymatic reactions are occurring at their maximum rate

microbes that grow between 25-40°C, around body temp which is 37°C

microbes that thrive in high temperatures, ~50°C or higher

microbes that thrive in low temperatures, ~15°C or lower

Infusion medium is soaked animal or plant materials in water that support the growth of microbes. Unknown amounts and cannot be reproduced. ex: Hay Infusion, BHI

Complex medium contain extracts of natural products like soybeans, beef, tissue, yeast cells, milk, etc. Unknown amounts but can be reproducedUsed for microbes that have multiple or unknown nutrient requirements ex: peptones, tryptones, casein, yeast and beef extract in TSA, PGS

Defined medium contain only chemically identified substances in known amounts and can be reproduced.
ex: GS

C, N, O, K, P, Na, S, Mg, Ca

Mn, Fe, Co, Cu, Zn, Mb

Chemoautotrophs and photoautotrophs both get their carbon source from carbon dioxide and chemoheterotrophs and photoheterotrophs both get their carbon from organic substances. Chemoheterotrophs and chemoautotrophs both get their energy from chemical substances while photoautotrophs and photoheterotrophs get their energy from light.

The speed of chemical reactions, the rate of bacterial growth, pigment production, total amount of growth are affected by temperature.

Psychrophiles thrive at low temperatures at 15°C or lower, mesophiles thrive between 25-40°C usually body temperature at 37°C, and thermophiles thrive at high temperatures at 50°C or higher

Aerobes- possess enzymes that allow them to utilize oxygen and neutralize toxic intermediate products formed during respiration process, must have oxygen to grow, oxygen is electron acceptor in ETC

Microaerophiles- need oxygen concentrations between 2-20% for growth

Facultative anaerobes- prefer to grow in the presence of oxygen because they have cytochromes, but will grow under anaerobic environments if a suitable substitute for oxygen is present like nitrate, can respire oxidatively or fermentatively

Anaerobes- oxygen in any content is toxic to microbe, no enzyme to neutralize toxic intermediates

stab with a needle into thioglycollate broth

Anaerobic jar, glove box, and thioglycollate tube.

resazurin is located throughout the tube

The function of thioglycollate is to create an oxygen gradient within a tube. top layer is aerobic, middle is microaerobic and the bottom is anaerobic. glucose is present as the carbon and energy source

The advantages of thioglycollate medium is that it's inexpensive and easy to transport, but difficult to obtain a true anaerobic environment.

The advantages of the anaerobic jar are it has lower oxygen concentrations and are pretty inexpensive, but must have a good seal on the jar, and still can be exposed to oxygen with transport of microbe to the jar.

The advantages of the glove box are that there is a lot of space, there is no oxygen present whatsoever but is expensive and takes up lots of room in the laboratory.

redox indicators detect the presence of oxygen. They are colorless when very little oxygen is present, but will turn red (resazurin) or blue (methylene Blue) is oxygen is present. determines how deep the oxygen has penetrated the media and how much is left anaerobic.

Anaerobes cannot live in the presence of oxygen because they lack the enzymes catalase or superoxide dismutase, that can break down the toxic intermediates of oxidative respiration.

organisms that require high temperatures for growth and thrive in heat

organisms will tolerate and survive high temperatures but will not be able to grow and reproduce at high temperatures.

binding of two adjacent thymine residues, prevents DNA replication, found in UV irradiation

chemotherapeutic substances used to inhibit or kill microorganisms, chemical control

the lowest concentration of a drug that prevents visible growth of the organism, units per ml

subgroup of antimicrobials which are biosynthesized by other microbes ex: penicillium, streptomyces, bacillus

inhibit growth of bacteria

destroy bacteria

zones of no growth around discs in Kirby Bauer disc diffusion method

minimal concentration of a antimicrobial required to kill the microorganism

It's necessary to control the growth of microorganisms because we must be able to combat adverse effects like contamination or disease.

Physical: heat, pasteurization, ultra-high-temperature (UHT) treatment, autoclaving, and UV light irradiation

Chemical: dyes, antimicrobial agents, antiobiotics

Thermophilic require high temperatures to grow and reproduce while thermoduric organism tolerate high temperatures but cannot reproduce in them

Pasteurization- high temperatures, kill non-sporeforming bacteria, destroys pathogens and reduces number of microbes to prolong shelf life (of milk), around 60-100°C, no use of pressure, thermoduric microbes can survive, thus must be refrigerated, no steam

UHT- exposes milk to extremely high temperatures fora brief period of time, can allow milk to be stored at room temperature at long periods of time.

Autoclaving- sterilization process, high heat, pressure, and steam to kill non-sporeforming and sporeforming cells, 121°C for 15 minutes with 15 pounds of pressure

Some microbes survive heat and UV radiation because of their spores or pigmented cells.

physical control, absorbed by purine and pyrimidine bases of DNA, prevents DNA replication, poor penetrating ability, growth around rim of plate because plastic can get in the way of UV light

Low concentrations of crystal violet are bacteriostatic for Gram (+) but not Gram (-) because of differences in cell wall structures (LPS of Gram negative)

Dyes and antimicrobials are both chemical agents of microbial growth control, but dyes are used for selective and differential media, usually don't kill microbes, but antimicrobials inhibit growth or kill microbes via cell wall synthesis, ribosomal action, protein synthesis, and nucleic acid synthesis

increasing concentrations of antimicrobials in test tubes, has a MIC, very sensitive and yields data for direct comparisons among antimicrobials, synergistic and antagonistic effects, possible to test 2 or more antimicrobials at the same time, but tedious, time-consuming, too expensive

involves a microtiter plate, a tray with many wells, varying concentrations are put in the wells, see growth in wells after incubation, has a MIC, fast specific information but expensive

most common, agar plates of increasing concentration of antimicrobial is used, multiple isolated of microbes can be tested at one time on a single plate, expensive, time consuming, takes up a lot of room

new diffusion test, plastic strip with a gradient of antimicrobial, U shape zone of inhibition, MIC at the apex of the U, inexpensive

single high potency discs are used for each antimicrobial, put on surface of plates, after incubation they are examined for zones of inhibition around the discs, susceptibility and resistance based on diameter of zone, has no MIC, highly standardized, Gram (+) = more susceptible than Gram (-) = more resistance

A large zone doesn't indicate usefulness because an antimicrobial will diffuse into the medium around the disc

Not always possible to relate in-vitro to treatments inside organisms because there are high complex interactions in the body, so could be affected by factors like immune response of host, inflammation, bacterial virulence, number of organisms present

cell wall synthesis

protein synthesis

protein synthesis

protein synthesis

cell wall synthesis

nucleic acid synthesis

protein synthesis

a control is a placebo to check the function of each component of the experiment to confirm that the reagent, microbe, or environment involved is functioning as expected.

Bacillus sp. and Clostridium sp.

barium sulfate nephelometer standards mimic density of various concentrations of bacteria growing in broth cultures.

0.5 McFarland Standard = 1.5 x 10^8 bacteria/ml

dilute concentrated cultures to standard concentrations for Kirby- Bauer

need fresh culture, add to sterile saline tube and add until densities match between sample and standard, mix frequently to compare, turbidity

How Does This Work?: stab with a needle to bottom of the tube and streak the slant in a zig-zag

Original Color of Medium: beige, tan, N/A

Microbes Selected For and Against: study Gram (-) organisms

Selective Agents (Inhibitors):

Differential Agents: fermentation of glucose, lactose, sucrose, production of gas, ferric ammonium citrate and sodium thiosulfate (produce hydrogen sulfide)

pH Indicator: phenol red

Interpretation of Results:
Red/Yellow = K/A = only glucose utilized, rapid depletion of glucose on slant so use peptones, releases ammonia, which is why alkaline slant is produced, aerobic

Yellow/Yellow = A/A = glucose, and lactose and sucrose utilized, a lot more lactose and sucrose so more acid is formed

Yellow/Black = A/A = glucose, lactose, sucrose, H2S production

Red/Black = K/A = glucose and H2S production

Red/Orange = K/NC = only peptones utilized, unable to ferment any CHOs

Black or Blue/Orange = K/NC = peptones utilized with pigment production

Gas formed: splitting of the medium, bubble formation, empty space below agar

H2S Production: black, yellow is beneath, anaerobic conditions, only seen ***********

plate that contains 30-300 colony forming units (CFU)

microbes which are not only commonly found in association with pathogenic species, but which are usually found in much higher numbers than pathogens ex: E. coli

spread plating 0.1 ml of water onto PCA and incubating to find CFU/ml

used to determine the concentration of heterotrophic organisms in water

Aseptically transfer 1.0 ml of sample to first tube in dilution series, with a new sterile pipette aseptically transfer 1.0 ml from that tube to the second tube, repeat this step.

techniques which repeat the same dilution through a series of tubes

Spread Plate: small volume of the sample is spread over entire agar surface

Membrane Filtration: necessary to count number of cells in a certain volume of liquid, force liquid through membrane filter and put on agar plate, small number of microbes

# CFU x FDF

...

it is important to determine the number of microbes because can contain bacteria, molds, yeasts and can question the safety of the food or water samples.

general environment from where the food was obtained, microbial content of food in unprocessed state, sanitary conditions during processing, adequacy of subsequent packaging and storing

Gram (+), nonmotile, aerobic, microaerophilic, facultative anaerobic rods or cocci

ex: Lactobacillus (probiotic), Listeria, Mycobacterium, Micrococcus, Streptococcus

Raw Milk = 100,000 CFU/ml
Pasteurized Milk = 20,000 CFU/ml
Pasteurized, Cultured Products = N/A

They are easier to detect than pathogenic organisms and when found, can assume that the water has been contaminated with fecal matter, potential for enteric pathogens in a water supply.

Escherichia coli

Wells: <1 fecal coliform/100 ml water
Public Distribution of Water: <1 fecal coliform/100 ml water
Swimming Areas: <235 fecal coliforms/100 ml water

<500 Total Heterotrophs/ml

microbes that will ferment lactose and produce gas within 48 hours

includes both environmental and fecal coliforms

coliforms originating from human colons like Escherichia coli

How Does This Work?: membrane filtration test will 100 ml of filtered water, placed on surface of agar plate, 24 hours at body temp

Original Color of Medium: maroon-red

Microbes Selected For and Against: detection of coliforms via membrane filtration, Gram (-) microbes

Selective Agents (Inhibitors): sodium desoxycholate and sodium lauryl sulfate, inhibit Gram (+) and non-enteric Gram (-)

Differential Agents: lactose
Basic Fuchsin and sodium sulfite to detect by products of metabolic processes, aldehyde compounds create red color

pH Indicator:

Interpretation of Results:
Strong Lactose Fermenter (E coli): dark red colonies with a green metallic sheen
Lactose Fermenters (Coliforms): red colonies
Non-Lactose Fermenters: light pink colonies

general, nutritional medium, grow organisms that usually live in a diluted environment, allow microbes to thrive, food and water samples

dilution factor inside the flask of the original sample used in the experiment. if not diluted, then SDF=1/1

volume of sample collected in the tube/volume of the sample transferred and volume of dilution

ex: 0.1ml/ (9.9ml+0.1ml) = ITDF=1/100

product of all ITDF up to the tube associated with the countable plate

how much of the sample is plated FOR THE COUNTABLE PLATE, always compared to 1 ml

ex: 0.1ml plated/1 = PDF=1/10

product of all the factors: SDF x TSDF x PDF

Too numerous to count colonies on plate, more than 300 colonies

mutations of DNA within the genes of a single individual

shuffling of alternate forms of genes/alleles that have accumulated over a population over several generations between two different chromosomes

the formation of an altered DNA sequence resulting from the exchanges of DNA from two different sources

changes in the sequences of the building blocks of DNA (nucleotides)

sometimes beneficial to microbe and organism- antimicrobial

involves the transfer of an extrachromosomal piece of DNA (F plasmid) between a donor and a recipient bacterium that are in direct contact with one another

more frequent in Gram (-) than (+)

may lead to antimicrobial resistance

donor bacterial strain comes into physical contact with a recipient strain and transfers genetic material

selective for metabolic pathways and antibiotic resistance

uptake of soluble, free/ naked chromosomal DNA by competent bacteria cells changing the genetic constitution (genome) of a bacterial cell

donor cell has DNA that recipient cell is lacking, acquires a new genetic property

selective for antibiotic resistance

transfer of genes from one bacterial cell to another by a bacterial virus, bacteriophage that inhabits at the donor bacterial strain

donor bacteria is infected by bacteriophage, bacteriophage injects its DNA into the host cell, breaks up bacterial chromosome, creates viral DNA, viral particle assembly, host bacteria lyses, new virus released to infect a recipient cell with mostly viral DNA, some donor bacterial DNA

selective for metabolic pathways

extrachromosomal genetic elements that encode a number of DNA transfer functions, possess transfer genes that specify and control the formation of pilus, substances to minimize donor-donor mating, transfer of plasmid or chromosomal DNA

"luxury" because often times code for products not essential for normal metabolism, usually antibiotic resistance or alternative metabolism

an extrachromosomal genetic structure that can replicate independently within a bacterial cell

ex: F plasmid- replication and number of conjugative plasmids
R plasmid- antibiotic resistance

extrachromosomal genetic elements that encode a number of DNA transfer functions

recipient bacteria that have taken up a plasmid, the original recipient parental strain which participated in conjugation and now has the F plasmid

free naked DNA transfered to a recipient cell

uptake of free nakes DNA into genome

bacterial viruses, also known as phages, only infect specific cells with receptors they can recognize

causes lysogeny, viral resting phase

recipient cells that are capable of incorporating free DNA, can become competent by changing environmental conditions

specific conditions are necessary to obtain maximum competence of recipient cell populations:condition of donor DNA, stage in growth cycle of recipient, nutritive value of medium, presence of cations

changing the environment that the recipient lives in to become a competent cell

a recipient cell that uptakes free naked DNA and integrates into its DNA genome

grow around transformants on a transduction experiment plate because there is not ampicillin surrounding transformant, so use that little space to grow and divide.

tiny colonies growing as satellites around larger colonies, larger colonies are transformed cells which are capable of producing beta-lactamase, which is an enzyme that destroys ampicillin around larger colony.

recipient cells that acquire a donor trait

the bacteriophage involved in transduction, transducing particle, doesn't contain a full copy of its own DNA, contains some bacterial DNA

virus is able to absorb and penetrate, but cannot progress further through the lytic cell

Viral resting phase, viral DNA is integrated into bacteria DNA and is duplicated when the bacteria duplicates its DNA, can continue indefinitely

viral DNA insert into a bacterial DNA genome during lysogeny

bacterial cell with the integrated virus prophage

viral reproducing phase, usually manifests itself, injected viral DNA takes over bacteria's protein manufacturing machinery and directs the synthesis and assembly of new viral particles until all phage particles are put together, then cell will lyse releasing new bacteriophage.

infecting virus that causes lytic cycle in host cell, doesn't cause lysogeny

virulent bacteriophage capable of infecting and lysing Salmonella

discrete circular clearing that form amidst a lawn of confluent bacterial growth as a result of the lytic response, areas of reproducing virus and dead bacteria

plaque stops growing when bacteria reach stationary growth because viruses cannot infect

results from the single initial infection which spreads outward concentrically, theoretically from one phage particle

a plate of confluent bacterial growth

structure: F+ (independent in cytoplasm), Hfr (part of bacterial cell), F' (detached from bacterial chromosome with some of its own genes)

Hfr = fastest

gene: responsible for the replication and number of conjugative plasmids produced

1. Use a MG/STR plate
2. streak the donor (ERW) down the middle of the MG/STR plate
3. streak the recipient (B380) across the middle of the MG/STR plate

The controls worked if there is only growth in the middle or a little bit to the right of the middle. The controls should not have grown on the plate because ERW- is susceptible to streptomycin (STR) and B380 is unable to grow without histidine.

The experiment worked if there was growth where the two microbes came in contact with each other in the middle of the plate. This means the his+ gene was conjugated from ERW to B380 so B380 could grow without the presence of histidine

Spontaneous mutants would have grown in another area besides growth on controls.

1. transfer all DNA to the experiment tube
2. Incubate on ice
3. Heat shock
4. Add 0.2 ml of LB Broth
5. Plate experiment DNA onto an LB/AMP Plate
6. Plate control DNA onto an LB Plate and an LB/AMP Plate

The controls worked if there was no growth on the LB/AMP Plate and confluent growth on LB Plate. Control 1 demonstrates the E. coli JA228 is a healthy microbe. Control 2 with no growth shows JA228 is susceptible to ampicillin so it won't grow when it is present.

The transformation worked on the transformation LB/AMP experiment plate if there is growth of transformants on the plate, who are ampicillin resistant. They grow and satellite colonies grow around the transformants in the space where no ampicillin is present.

Look for spontaneous mutants on the Control 2 LB/AMP Plate because they would be ampicillin resistant, so they would grow when E. coli JA228 wouldn't.

1. Obtain 1.1 ml of Salmonella typhimurium
2. Transfer 0.3 ml of S. typhimurium as bacterial control
3. Transfer 0.1 ml of P-22 bacteriophage to the remaining 0.8 ml of Salmonella as experiment tube.
4. Incubate 3 tubes, experiment, viral and bacterial control for 15 minutes.
5. Plate 0.1 ml of the bacterial control to an MG plate labeled bacterial control 2.
6. Place 0.1 of the bacterial control onto an MG/TRP plate labeled bacterial control 1.
7. Transfer experiment to two MG plates, experiment 1 and 2.
8. Place the viral control onto an MG plate, viral control plate

The controls worked if viral control plate had no growth because viruses are dead. There should be confluent growth on bacterial control 1 because it is the donor. It has trp+ so it can grow and use its own nutrients and tryptophan to live. Bacteria control 2 should have no growth because needs tryptophan to live and it is absent from the environment.

Transductant will have picked up the trp+ gene from the phages and how has own nutrients of tryptophan to grow, so there was lots of growth on the plate.

Look for on bacterial control 2 where there should be no growth, if there are spontaneous mutants they would be tryp+ and be able to grow.

replication with a host:

replication without a host:

transfer of DNA between bacteria: viral attachment of bacteriophage to bacteria cell and injected in or uptake via pilus or naked DNA

harmful or beneficial to bacterial host?:

physical manifestations if harmful to host: bacteriophage lyse the cell in the end

physical manifestations if helpful to host: can transfer genes that are helpful for survival of bacteria

types of DNA transferred:
conjugation- plasmids (F plasmid)
transformation- naked chromosomal DNA or plasmids
transduction- mostly viral DNA, some bacterial DNA is transferred

how DNA is transferred:
conjugation- plasmid via pilus
transformation- taken up into cell and incorporated into recipient DNA genome
transduction- transferred via bacteriophage from donor to recipient cell

the physical manifestation is the lyse of the bacteria cell and the release of bacteriophage to infect more bacterial cells

steps involved:
lysogeny-attachment, penetration, resting phase
lytic cycle- attachment, penetration, synthesis, lyse and release

immediate or later:
lysogeny- later
lytic- immediate

physical manifestations of phage:
both end in the lysing of the cells, but at different times

type of virus:
bacteriophage

interaction with host cell:
both attach, penertrate
lysogeny- incorporate viral DNA into bacterial DNA and duplicates over time
lytic- takes over bacteria proteins for manufacturing and creates many new viral particles

Plaques develop when viruses lyse bacteria and go on to infect other bacteria

they don't develop in plaque assay procedure because bacteria stop growing and cannot be affected by virus

Bacteriophage need to reproduce in host cell so they can use the mechanisms inside the bacteria to create more viral DNA to create more viruses. Require metabolic machinery, obligate intracellular parasites

DILUTING THE VIRUS
1. Transfer all 0.5 viral culture to the TPB tube (X 10 ^-1)
2. Transfer 0.5 of X to Y (10^-2) tube
3. Transfer 0.5 of Y to Z (10^-3) tube
BACTERIA CONTROL
1. Add 0.3 ml E. coli to soft agar, then pour into a plate.
VIRAL CONTROL
1. Add 0.1 ml of TPB tube X to the soft agar, plate.
DILUTED VIRAL EXPERIMENT
1. Add 0.3 ml E. coli B and 0.1 ml virus, either X,Y,or Z into the soft agar and plate.

count number of countable PFUs on a plate

PFU/ ml = number plaques x (1/FDF)

actual genes the microbe posses

observable characteristics or traits presented by the microbe

microbe has non-functional arginine gene and will need arginine provided in the medium to be able to grow

microbe has a functional arginine gene and will not need a source of arginine to grow

microbe has a non-functional histidine gene and will need histidine provided in the medium to be able to grow

microbe has a functional histidine gene and does not need a source of histidine to grow

microbe has a non-functional tryptophan gene and will need tryptophan provided in the medium to grow

microbe has functional tryptophan gene and doesn't need a source of tryptophan to grow

microbe is sensitive to ampicillin and will not grow in any medium which contains this antibiotic

microbe is resistant to ampicillin and is capable of growing in the presence of ampicillin

microbe is sensitive to streptomycin and will not grow in any medium which contains this antibiotic

microbe is resistant to streptomycin and is capable of growing in the presence of streptomycin

donor cells, capability of making all of the essential elements necessary for growth and can grow on a medium which is devoid of extra growth factors

ex: Minimal Glucose

recipient cells, mutant cell that is lacking a growth factor that is essential for growth

a medium that contains a certain amino acid that can be selective for some microbes, must have genes for synthesis

enzyme secreted from E. coli JA228 amp (r) that destroys the surrounding ampicillin

Bacteriophages that have virus heads packed with bacterial host DNA, cannot produce any more viruses but can still infect a bacterial cell and inject bacterial host DNA into bacteria cell, which could potentially replace DNA inside host genome

a mutant cell which has undergone another mutation where the second mutational event reverses what happened in the initial mutation

How Does This Work?: selective, only microbes that can synthesize their own nutrients can grow on this plate

Original Color of Medium: tan, N/A

Microbes Selected For and Against: must be able to synthesize whatever nutrients are lacking in the medium, prototrophs and hardy microbes

Nutrients: glucose, N, salt, P

Selective Agents (Inhibitors): N/A

Differential Agents: N/A

pH Indicator: N/A

Interpretation of Results:
growth or no growth based on genes present

How Does This Work?: inhibits protein synthesis via streptomycin

Original Color of Medium: tan, N/A

Microbes Selected For and Against: microbes that are streptomycin resistance, prototrophs

Nutrients: glucose, N, salt, P, streptomycin

Selective Agents (Inhibitors): streptomycin

Differential Agents: N/A

pH Indicator: N/A

Interpretation of Results:
growth or no growth based on if the microbe has streptomycin resistant gene or not

How Does This Work?: selective, only microbes that can synthesize their own nutrients can grow on this plate

Original Color of Medium: tan, N/A

Microbes Selected For and Against: must be streptomycin resistant and supports growth of histidine auxotrophs

Nutrients: glucose, N, salt, P, streptomycin and histidine

Selective Agents (Inhibitors): streptomycin, histidine

Differential Agents: N/A

pH Indicator: N/A

Interpretation of Results:
growth or no growth based on genes present, his- and str (r)

How Does This Work?: selective, only microbes that can synthesize their own nutrients can grow on this plate

Original Color of Medium: tan, N/A

Microbes Selected For and Against: supports auxotrophic tryptophan (trp-) microbes

Nutrients: glucose, N, salt, P, tryptophan

Selective Agents (Inhibitors): N/A

Differential Agents: N/A

pH Indicator: N/A

Interpretation of Results:
growth or no growth based on genes present, growth if trp-

F+ cell: a donor cell carrying the F plasmid, which is not attached to the bacterial chromosome

F- cell: a recipient cell lacking the F plasmid

Hfr cell: a bacterial strain that exhibits a high frequency of gene transfer and recombination during a mating process. The F plasmid DNA is part of the bacterial chromosome, usually from a F+ cell

F' cell: a bacterial strain that has the F plasmid not integrated in the bacterial chromosome, obtained a piece of bacterial chromosome DNA, derived from a Hfr cell, when plasmid is removed from the chromosome

prototroph

MG Plate: growth because have F' plasmid with his+

MG/STR: no growth because str (s)

MG/STR/HIS: no growth because str (s)

auxotroph

MG: no growth because his- and medium contains no histidine

MG/STR: no growth because his- and medium contains no histidine

MG/STR/HIS: growth because str (r) and medium contains histidine

MG: growth because has F' plasmid with his+

MG/STR: growth because str (r) and has F' plasmid with his+

MG/STR/HIS: growth because str (r), but could lose plasmid if in histidine medium

auxotroph

MG: no growth because is arg- and medium contains no arginine

MG/STR/ARG: growth because str (r) and medium contains arginine

LB: growth because medium has all essential amino acids

LB/AMP: no growth because amp (s)

MG: growth because strp (r) and amp (r), medium contains arginine

LB: growth because medium has all the essential amino acids

LB/AMP: growth because is amp (r) and medium has all essential amino acids

prototroph

MG: growth because is trp+

auxotroph

MG: no growth because trp- so needs tryptophan to grow

MG/TRP: growth because medium contains tryptophan

MG: growth because has new trp+ from P-22

MG/TRP: growth because has new trp+ from P-22, so TRP in medium isn't needed but won't impact growth

ERW can grow on the donor line streak if it spontaneously mutates and becomes streptomycin resistant.

B380 can grow on the recipient line streak it is spontaneously mutates and becomes his+, would become a revertant

Spontaneous mutants that are ampicillin resistant can grow when JA228 is plated

name of antimicrobial resistance acquired: amp (r)

type of DNA where resistance resides: plasmid is naked DNA

what additional gene has been acquired: beta-lactamase gene

gene product: beta lactamase enzyme

The spontaneous mutant would be the revertant Salmonella typhimurium. The trp- gene has mutated back again to trp+ and can grow on the plate

1. Adsorption- host specific bacteriophage attachment to the bacteria cell
2. Penetration- viral nucleic acid enters bacterial cell
3. Synthesis- virus takes over bacterial replication mechanisms and makes viral nucleic acids and proteins
4. Assembly- viral proteins and nucleic acids are put together to create new viruses
5. Release- cell is lysed and new viruses are released

1. Adsorption- virus attaches to bacteria cell, host specific
2. Penetration- viral nucleic acid enters bacterial cell
3. Integration- viral DNA is inserted into bacterial chromosome and becomes part of the chromosome rather than causing viral synthesis, virus can stay integrated like this indefinitely until some event occurs which activates, forces out the virus
4. Induction- when the viral DNA removes itself from the bacterial chromosome, when cell is damaged or about to die
5. Synthesis- makes viral proteins and nucleic acids
6. Assembly- creation of viruses
7. Release- cell is lysed and release of viral progeny

microbes which are only associated with the body for a short time due to variations in diet, health, etc.

microbes comprising relatively permanent, natural residency and are constant over time

microbes able to cause disease, usually held in check by competitive and beneficial normal microflora

changes in the normal balance of indigenous microbes because analagous disturbances that are already present can cause disease state

ex: Candida albicans

Candidas albicans lives in competition in the intestine, but if broad spectrum of antimicrobials are used, they will wipe out many normal microflora. Candida albicans is eukaryotic so it will survive since it's resistant to bacterial antibiotics

also known as Neisseria meningitidis, live on the mucous membranes inside nasopharynx, causes bacterial meningitis via respiratory secretions

healthy people who harbor potentially pathogenic organisms like S. aureus

hospital-acquired infection, can occur through aerosols in a sneeze, touching nose or face, secondhand disease, usually occurs in immunocompromised individuals

increased resistance due to transfer of plasmids

Candidas albicans

Actinomycetes, Bacteriodes sp., Bifidobacterium sp., Clostridium sp., Enterococcus sp., Fusobacterium sp., Escherichia coli, Lactobacillus sp., Peptostreptococcus sp., Staphylococcus sp., Streptococcus sp., Veillonella sp.

many do not grow on ordinary media, Streptococcus mutans, Streptococcus sanguis

Actinomycetes, Bacteriodes sp., Candida albicans, Fusobacterium sp., Lactobacillus sp., Moraxella catarrhalis, Neisseria sp., Staphylococcus sp., Streptococcus sp., Veillonella sp.

Neisseria meningitidis, meningococcus, Staphylococcus aureus

Corynebacterium sp., Haemophilus sp., Lactobacillus sp., Moraxella catarrhalis, Neisseria sp., Staphylococcus sp., Streptococcus sp., Streptococcus pneumoniae

aerobes and facultative aerobes

Candida albicans, Corynebacterium sp., Micrococcus sp., Propionobacterium sp., Staphylococcus sp., Streptococcus sp.

name: Candida albicans

disease: Candidiasis

what happens in body to cause disease: antimicrobials wipe out normal microflora, eukaryotic so can take over an cause an intestinal infection

relationship between human and microbe: usually outcompeted by normal microflora, but can be an opportunistic microbe

name: Streptococcus mutans

disease: dental cavities

place of adhesion: S. sanguis attach to tooth surface via fimbriae and then S. mutans can attach to S. sanguis

relationship between human and microbe: fermentation producing acid extracts Ca from tooth enamel causing dental carries

name: S. aureus

disease: nosocomial infection

type of patient susceptible: immunocompromised, those already in the hospital

periodic screening

sampling the anterior nares with a swab, incoulation of selective, differential agar and general purpose agar, interpretation of the medium, treatment of the carrier

seldom done unless there is an outbreak

physical and chemical barrier

washing doesn't remove microbes because of the topography of the skin with the layer of body oils present

skin asepsis- strong germicides (betadine) surgical scrubbing procedures

normal flora on skin outcompete pathogenic microbes on skin

If they were gone, they couldn't outcompete pathogens and would enter through breaks, cuts, and scratches in the skin

beneficial microbes keep pathogenic bacteria in check on the skin

to determine the correct treatment to give, don't give a bacterial antimicrobial if it is a eukaryote because won't kill the microbe and will allow microbe to flourish because it won't have to compete with other bacteria since they're all gone

found in the production of dental carries, attach more readily to tooth surface if S. sanguis is present

acid production from fermentation, extracts Ca from tooth enamel causing dental caries, tooth decay

hemolysins

BAP: Alpha, green halo, partial lysis via hemolysins

found in the production of dental carries, produces fimbriae that attach to the surface of the tooth

can produce gingival (gum) abscesses

hemolysins

BAP: Alpha, green halo, partial lysis of cells

Gram (+) purple rods

Catalase: (-)
forms small, pinpoint colonies on agar
normal oral, intestinal and vaginal flora
helps keep foreign microbes in check

TJ: pinpoint colonies

Gram (-) diplococci, kidney bean shaped

Oxidase: (+)
causes meningitis

found in the nasopharynx

enterohemorrhagic E. coli

Gram (-) rod

Oxidase: (-)
additional genetic information that codes for toxins, food poisoning

How Does This Work?: isolate and different oral streptococci

Original Color of Medium: light blue

Microbes Selected For and Against: selected for Gram (-) rods and streptococci

Selective Agents (Inhibitors): crystal violet, potassium tellurite, inhibit Gram (+)

Differential Agents: trypan blur, tellurite, glucose, sucrose

pH Indicator: N/A

Interpretation of Results: N/A

How Does This Work?: color change based on metabolism and growth based on ability to live in high salt concentrations

Original Color of Medium: pink/orange

Microbes Selected For and Against: bacteria that can grow in high salt concentrations, screening for S. aureus, mannitol fermenter or peptone fermenter based on color of the media

Selective Agents (Inhibitors): 7.5% NaCl, selects for halotolerant bacteria

Differential Agents: mannitol, fermented to produce acidic by-products or peptone breakdown to form alkaline products

pH Indicator: phenol red

Interpretation of Results:
growth: halotolerant
no growth: not halotolerant
mannitol fermenter: growth, yellow colonies with yellow surrounding media
non-mannitol fermenter: growth, red colonies with red surrounding media

How Does This Work?: isolation and identification of Candida albicans and Lactobacillus sp., oral microbes

Original Color of Medium: brown

Microbes Selected For and Against: selected for specifically Candida albicans and Lactobacillus sp.

Selective Agents (Inhibitors): tomato juice, low pH of the medium

Differential Agents: N/A

pH Indicator: N/A

Interpretation of Results:
Candida albicans: creamy opaque colonies
Lactobacillus sp: pinpoint colonies

ability to cause a disease, yes or no question

the degree of pathogenicity, based on virulence factors, how able microbes are to evade immune system

subdivision of virulence
production of toxic compounds by an organism

subdivision of virulence
ability of an organism to colonize a host, resist host defense mechanisms and spread

engulfment by host white blood cells

extracellular enzyme produced by S. aureus to avoid phagocytosis, converts fibrinogen to fibrin, causes coagulation

form fibrin walls around the staphylococcal lesion, layer of fibrin surround each staphylococcal cell, resist host defense mechanisms, anti-phagocytic properties

separate S. aureus from S. epidermidis

two forms, bound and free

"clumping factor", remains attached to the cell surface, when S. aureus is mixed with rabbit plasma, fibrin is formed around the cells causing them to clump

if negative for slide test, do tube test

BOUND COAGULASE= SLIDE TEST

present in cell-free filtrates, will cause a small tube of rabbit plasma to coagulate

FREE COAGULASE = TUBE TEST

proteins produced by bacteria that cause animal cells to lyse, usually rbc (erythocytes), release hemoglobin

vary in enzymatic properties,sensitivity to environmental changes, immunological traits, types of cells that they lyse, temperature

similar to hemolysins, proteins produced by bacteria the kill animal cells, lyse rbc, immune cells

proteins that are made in the bacterial cell and secreted into the surrounding medium

ex: hemolysins

toxic proteins excreted by bacteria that enhance the virulence of many potentially pathogenic microbes

ex: hemolysins

major hemolysin produced by S. pyogenes

host responds to SLO by producing antibodies that cause heart tissue damage

inactivated by oxygen

major hemolysin produced by S. pyogenes

not inactivated by oxygen

relatively thick layer of high molecular weight polysaccharides that surround the entire microbial cell, anti-phagocytic properties

ex: S. pneumoniae

color, can have virulence factors

binds iron

blue pigment found in Pseudomonas aeruginosa

stimulate the release of interleukin-8

green fluorescent pigment found in Pseudomonas aeruginosa

siderophore that binds iron

same as pyoverdin

red pigment found in Pseudomonas aeruginosa

brown pigment found in Pseudomonas aeruginosa

1. Transfer one drop of distilled water and add a small portion of microbe to the water and mix.
2. Transfer one drop of rabbit plasma to slide and add a small portion of microbe to the plasma and mix

INVALID: microbe clumps in water and rabbit plasma
POSITIVE: microbe clumps only in rabbit plasma
NEGATIVE: microbe does not clump in either, perform tube coagulase test to confirm

ex: (+)= S. aureus
(-) = S. epidermidis, S. saprophyticus

1. Transfer 2-3 colonies into the rabbit plasma tube
2. Cover and incubate

POSITIVE: clot formation, solid mass
NEGATIVE: liquidy, viscous

ex: (+) = S. aureus
(-) = S. epidermidis, S. saprophyticus

similarities: SLO and SLS both are hemolysins in S. pyogenes, lyse blood cells, alter membrane permeability, disrupt sterols in the tissue cell membrane

differences: SLO- host responds by making antibodies that cause heart tissue damage, inactivated by oxygen

SLS- not inactivated by oxygen

alpha: green halo, partial lysis of rbc via hemolysins, ex: Streptococcus pneumoniae
beta: tan halo, complete lysis of rbc via hemolysins, ex: Streptococcus pyogenes, Streptococcus agalactiae
gamma: no lysis of rbc via hemolysins

Differential because growing in sheep's blood. Identifies streptococci via its hemolytic action on the red blood cells in the medium

The function of the capsule is to create anti-phagocytic properties where they evade immune responses because host cells don't recognize cell as foreign

what are they made of, what they do, how do they evade the immune system

Coagulase: extracellular enzyme, avoids phagocytosis, convert fibronogen to fibrin to form around staphylococci lesion and cells

Hemolysins: extracellular proteins produced by bacteria that lyse animal cells, usually rbc, exotoxins, enhance virulence, produce antibodies and lysis of cells

Capsule: thick layer of polysaccharides surround whole cell, anti-phagocytic properties

Pigments: virulence factors like release of interleukin- 8 and siderophore to bind to iron, pyorubin, pyomelanin, pyocyanin, pyoverdin

Group A streptococci, Gram (+) cocci chains

BAP: beta hemolytic

virulence factors: streptolysin O and S- alter membrane permeability, damage sterols in tissues

cause strep throat, wound infections, kidney disease, rheumatic fever, flesh eating bacteria

Group B streptococci, Gram (+) cocci chains

Catalase: (-)

BAP: beta hemolytic

causes neonatal septicemia, form of meningitis

Gram (+) cocci clusters

Catalase: (+)

Coagulase: (-)

usually on normal skin microflora

Gram (+) cocci clusters

Catalase: (+)

Coagulase: (-)

causes UTIs

Group A streptococci, Gram (+) cocci chains

Catalase: (-)

BAP: Alpha hemolytic

normal throat flora, cause endocarditis, dental caries

capsule present outside bacterial cell

no pigment on TECH plate tube, still tan/ N/A color medium

How Does This Work?: ability of a microbe to clot plasma using coagulase, convert fibrinogen to fibrin, clumping factor, bound coagulase

Original Color of Medium: N/A

Microbes Selected For and Against: differentiate within Staphylococcus, S. aureus from non-pathogenic staphylococcus

Selective Agents (Inhibitors): N/A

Differential Agents: rabbit plasma

pH Indicator:N/A

Interpretation of Results:
invalid: microbe clumps in both water and rabbit plasma
positive: microbe clumps in rabbit plasma only
negative: microbe clumps in neither water nor rabbit plasma, perform tube coagulase test to confirm the lack of coagulase in microbe

How Does This Work?: ability of a microbe to clot plasma using coagulase, convert fibrinogen to fibrin, free coagulase

Original Color of Medium: clear liquid

Microbes Selected For and Against: differentiate within Staphylococcus, S. aureus from non-pathogenic h

Selective Agents (Inhibitors): N/A

Differential Agents: Rabbit plasma

pH Indicator: N/A

Interpretation of Results:
positive: solid formation in tube, clot formation
negative: liquidy and viscous

How Does This Work?: grows pathogenic bacteria, should be compared to BAP plate

Original Color of Medium: brown, chocolate colored

Microbes Selected For and Against: grow fastidious, pathogenic strains of Gram (-) organisms ex: Neisseria and Haemophilus

Nutrients: hemoglobin (X factor), NAD+ (V factor), general nutritional medium

Selective Agents (Inhibitors): N/A

Differential Agents: N/A

pH Indicator: N/A

Interpretation of Results:
flat grey colonies- Haemophilus
pulvinate, convex tan colonies- Neisseria

How Does This Work?: enhances ability of Pseudomonas aeruginosa to produce pyocyanin, inhibits other pigments

Original Color of Medium: tan

Microbes Selected For and Against: Pseudomonas aeruginosa

Selective Agents (Inhibitors): Magnesium chloride, potassium sulfate, low levels of phosphate to inhibit fluorscein production

Differential Agents: N/A

pH Indicator: N/A

Interpretation of Results:
Positive: blue green color is seen in the medium surrounding colonies
Negative: no blue green color in medium is seen

may seen pyomelanin (brown, grayish with pyocyanin), or pyorubin (red, purpleish with pyocyanin)

How Does This Work?:

Original Color of Medium:

Microbes Selected For and Against:

Nutrients: gelatin (movement with flagella), glucose (fermentable CHO for function of decarboxylate enzymes), ornithine (decarboxylate ornithine), tryptophan (production of indole via hydrolysis of tryptophan)

Selective Agents (Inhibitors): gelatin

Differential Agents: Tryptophan, add Kovac's reagent, ornithine

pH Indicator: bromcresol blue

Interpretation of Results:
Motility:
Positive: turbid, bottle brush pattern OR very turbid
Negative: clear tube, growth only on stabline

Indole:
Positive: Kovac turns cherry red
Negative: Kovac remains yellow-green

Ornithine:
Positive: bottom of MIO tube is purple
Negative: top of the tube is purple, bottom tube is yellow or entire tube is yellow

How Does This Work?: determines ability of microbe to hydrolyze glycoside and esculin to esculetin and glucose in the presence of bile, bile tolerance

Original Color of Medium: tan/beige/ N/A color

Microbes Selected For and Against: group D streptococci confirmation

Nutrients: glycoside, esculin

Selective Agents (Inhibitors): N/A

Differential Agents: N/A

pH Indicator: N/A

Interpretation of Results:
(NG/Unk): no growth is seen on streak line, reaction to esculin is unknown
(+/-): growth is seen, but medium is tan, no reaction to esculin
(+/+): growth is seen on streak, medium is brown/black

How Does This Work?: compare at a 0.5 McFarland Standard, streak a MH plate, place a Novobiocin disk in the middle of the plate, zone of inhibition is measured

Original Color of Medium: red

Microbes Selected For and Against: differentiate between Staphylococcus saprophyticus and Staphylococcus epidermitidis

Selective Agents (Inhibitors): Novobiocin disk

Differential Agents: N/A

pH Indicator: N/A

Interpretation of Results:
S. saprophyticus- <16mm zone of inhibition, resistant to novobiocin
S. epidermitidis- >16 mm zone of inhibition, sensitive to novobiocin

How Does This Work?: if produces enzyme gelatinase

Original Color of Medium: N/A color

Microbes Selected For and Against: microbes that have gelatinase enzyme

Selective Agents (Inhibitors): gelatin

Differential Agents: N/A

pH Indicator: N/A

Interpretation of Results:
Weak Positive: test tube liquefies and doesn't completely resolidify
Positive: test tube liquefies and remains a liquid
Negative: test tube liquefies and resolidifies

inoculate by melting media and then inoculate via loop

How Does This Work?: add latex/ antisera to heavily inoculated drop, look for agglutination

Original Color of Medium: blue from latex/antisera

Microbes Selected For and Against: distinguish between A, B, C, D, F, G streptococci

Selective Agents (Inhibitors): N/A

Differential Agents: latex/antisera

pH Indicator: N/A

Interpretation of Results:
Positive: Agglutination occurs, antigen corresponding with antisera
Negative: no agglutination occurs, microbe doesn't have antigen corresponding with antisera

How Does This Work?: determine if microbe is able to utilize carbon of sodium citrate as sole source of carbon for metabolism

Original Color of Medium: green

Microbes Selected For and Against: differentiation of Gram (-) rods

Nutrients: inorganic ammonium salts for nitrate sole source

Selective Agents (Inhibitors): N/A

Differential Agents: sodium citrate and ammonium salts

pH Indicator: bromthymol blue

Interpretation of Results:
positive: growth is seen, color is noted as blue but not needed
negative: no growth is seen, may be green still but color is not as important

blue: alkalinity products of carbonates and ammonia because sole nitrate and carbon sources

filamentous fungi, multicellular microbes, tend to form loose, fluffy colonies with aerial hyphae

filaments that make up fungal growth can be septate with cross walls or aseptate with no cross walls

long filaments which extend up into the air above the agar surface, differentiate into conidia or spore

unicellular microbes that form compact colonies resembling bacterial growth, do not form true hyphae

fungi exist in only one form, monomorphic yeast or mold

ex: Cryptococcus neoformans- monomorphic yeast
ex: Aspergillus sp., Rhizopus sp., Penicillium sp., exist in filamentous form- monomorphic molds

ex: Penicillium marneffei- grows in mold form in the environment and converts to a yeast form when infecting a human host, causes pneumonia

ex: Candida albicans- grows in a variety of forms like buds (blastoconidia), pseudohyphae

buds, conidia formed by the budding process

elongated blasoconidia and hyphae, elongated buds/blastoconidia

hyphae, asexual spores of filamentous fungi as borne on conidiophores

hyphae

producing structures of conidia

can be branched or unbranched

producing structures of spores, the stalk

sac like, mushroom top structure in rhizopus at the end of sporangiophores

the spores within the sporangium

reproductive structures, on aerial hyphae, asexual fruiting bodies

small, single celled conidia

large, multi celled conidia

Candida albicans forms a biofilm over surfaces of involved tissues in candidiasis

1. Insert needles into sand and vesphene
2. cut a portion of growth and add to slide
3. drop of Lactophenol Cotton Blue
4. tease the colony fragment, place a coverslip over it and seal with clear nail polish

1. place LPCB onto a slide
2. take a piece of scotch tape and press firmly onto surface growth of the fungi
3. pull off, tape to slide with LPCB

1. cut a piece of agar and put onto the glass slide in the petri dish
2. pick out a colony of mold from surface of agar and aerial hyphae
3. heat the coverslip and place on the agar surface until hear a sizzle noise
4. a little bit of water on the bottom of the petri dish

Lactophenol Cotton Blue, used to kill fungal cells but keeps them in their same spatial arrangement, stains chitin in cell walls to better see the fungus

a new daughter cell is produced as an outgrowth of the parent cell to create blastoconidia

hyphal extension produced from yeast cells upon germination, tube like projection, used to identify Candida albicans

surface pigmentation of a fungi

udnerneath the plate pigmentation of the fungi

Advantages: can get fruiting bodies and aerial hyphae, inexpensive, quicker
Disadvantages: harder to recognize spatial arrangement

Advantages: quick, easy, inexpensive
Disadvantages: only get aerial hyphae

Advantages: see all parts of the fungi, top and bottom
Disadvantages: expensive and time consuming

white stalks with green head, lollipop, won't fill the whole plate

conidiophore looks like a q-tip, unbranched conidia, septate hyphae

green velvet appearance, no stalks, light green, distinct border, won't fill the plate, yellow water soluble pigment

conidia are branched, look like skeleton hands, septate hyphae

large filaments, fill the whole plate with aerial hyphae, produce black around the outer edge of the plate, which are the fruiting bodies

is a sporangiophore, spore inside little sporangiospore cap, has rhizoid, root-like structures, aseptate hyphae

look like a turnip, with septate hyphae

reproduce by budding and will not produce germ tubes

its dimorphic, meaning it can change its form depending on the growth or environmental conditions

streak heavily for confluency to starve microbes leading to the production of endospores, general nutritional growth

X pattern, waves of growth over entire surface of the plate

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